Analysis of Clostridium botulinum Serotype E Strains by Using Multilocus Sequence Typing, Amplified Fragment Length Polymorphism, Variable-Number Tandem-Repeat Analysis, and Botulinum Neurotoxin Gene Sequencing

Abstract

ABSTRACTA total of 41Clostridium botulinumserotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin (bont) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinaserarA, involved withbont/Einsertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of thebont/Egene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, orbontgene sequencing were further examined using three VNTR regions. Both intact and splitrarAgenes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) theC. botulinumserotype E strains result from the targeted insertion of thebont/Egene into genetically conserved bacteria and (ii) recombination events (not random mutations) withinbont/Eresult in toxin variants or subtypes within strains.